CNTNAP4 Functions as a Specific Receptor of NELL-1 Affecting Osteoblastic Differentiation Chen-Shuang Li1,2, Xinli Zhang1, Yan-Heng Zhou2, Zhong Zheng1, Chia Soo3, Ting Kang1 1Division of Growth and Development and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, Los Angeles, CA USA, 2Department of Orthodontics, School and Hospital of Stomatology, Peking University, Beijing, China, 3UCLA Division of Plastic Surgery and Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA Introduction The NELL-1 gene was identified upregulation in craniosynostosis (CS) patients within prematurely fusing sutures (Ting et al., 1999). Transgenic Nell-1 overexpression mice exhibit gross bone overgrowth and a CS- like phenotype (Zhang et al., 2002). In contrast, Nell-1 deficient mice result in major skeletal anomalies with reduced bone formation (Desai et al., 2006; Zhang et al., 2012). Subsequently, NELL-1 was approved as a novel osteogenic protein inducing regeneration of bone in multiple animal models (Cowan et al., 2012; Kwak et al., 2013; Zhang et al., 2011a). To understand the mode of action of NELL-1’s osteogenic effects, several studies have been taken to indentify the potential cell membrane associated receptor(s) or interactive proteins of NELL-1. Recently, the intracellular molecule, apoptosis-related protein 3 (APR3) (Zou et al., 2011), was identified to physically bind to NELL-1 with only modest functional alteration of NELL-1’s ostegenic effects. Integrinβ1 was also found to bind with NELL-1 to mediate osteoblastic cell adhesion (Hasebe et al., 2012a; Shen et al., 2012). Generally, the Integrins are adhesion receptors for an enormous ligands population (Goessler et al., 2008), and not specific to NELL-1. Despite these accumulative data, the specific cell surface receptor(s) of NELL-1 responsible to its osteogenic effects hasn’t been identified yet. Materials and Methods 1. T7 human brain cDNA phage library construction and biopanning As NELL-1 is highly expressed in brain tissue (Nakamura et al., 2012; Ting et al., 1999), T7Select Human Brain cDNA Library was used to construct T7 phages library. For the maximal exposure of NELL-1 to its binding partners during biopanning, HIS-Select Nickel Magnetic Agarose Beads were selected to immobilize his- tagged NELL-1. An aliquot of the amplified phages was incubated with his-NELL-1 coated beads for 4 rounds of bipanning screen. The phages bound to his-NELL-1 coated beads were eluted, and 100 plaques were selected to amplify the phage DNA by PCR. The sequences larger than 500 bp were sequenced and analyzed (Fig. 1). 2. Dissociation constant (Kd) Fig 1. Diagram of phage biopanning by using His-NELL-1 coated magnetic beads. ELISA to confirm specific phage cDNA library was constructed from human brain mRNA and packaged with T7 phage. binding affinity The library was probed by his-NELL-1 coated magnetic beads. After 4 round of 10ug/ml of rhNELL-1 or short biopanning, most of the non-specific binding phages were washed off; the remaining phages were considered binding candidate and amplified the cDNA inserts by PCR using NELL-1 was used as bait to coat specific primers for T7 phage vector. PCR products over 0.5 kb were sequenced and the ELISA plate. After removed analyzed. unbounded bait, the plate was blocked with 3% non-fat milk. 100µl/well diluted phage lysate (1X104 – 1X1010 phages/ml) was added to the coated plate and incubated at 37 for 1 hour. Then, the unbound phages were washed out thoroughly, and T7 Tail-Fiber Monoclonal Antibody was added for 1 hour incubation. The HRP conjugated anti-mouse secondary ℃ antibody was added to the plate. For HRP Table 1. List of cell types used for Cntnap4 expression. detection, TMB substrate was used, and absorbance was read at 450nm. The T7 phages without insert were used as negative control, and each concentration of phage was triplicate. 3. Realtime-PCR of Cntnap4 gene expression of cell lines and primary cells 12 types of cell lines and primary cells (Table 1) which have significant response to NELL-1 were cultured under standard condition. When the cells reached subconfluence, RNA was isolated using standard protocol. And Cntnap4 gene expression level was detected by realtime-PCR. Table 2. List of rhNELL-1 binding candidate screened by 4. Pull-Down and Co-immunoprecipitation phage biopanning. Pull-Down PolyHis Protein : Protein Interaction Kit (Pierce) was used for Pull-Down assay. In brief, prey protein was isolated from MC3T3 or NMCC, and applied to the spin column which had pre-incubated with his-NELL-1 as bait protein. The bait-prey complex was eluted for SDS-PAGE. For CO-IP assay, MC3T3 or NMCC are trypsinized, suspended in PBS, and incubated with NELL-1. BS3 was added to the cells as crosslinker. Proteins were isolated by adding cold RIPA lysis buffer to the collected cells, and incubated with anti-NELL-1 antibody coated agarose beads. The his-NELL-1 – candidate receptor(s) complex was eluted for SDS- PAGE. 5. Small hairpin RNA transfection MC3T3 cells were transfected with Cntnap4 shRNA or nontarget control shRNA. The positive transfected colonies were selected by Puromycin, and validated by Cntnap4 mRNA expression level to establish Cntnap4 KD (knockdown) cell line. 6. Immunocytochemistry and confocal microscopy Serum-starved MC3T3 cells were treated with PBS or 500ng/ml rhNELL-1 for 30min, and fixed with ice-cold methanol. The fixed cells were blocked with 3% bovine serum albumin and incubated with anti-CNTNAP4, anti- NELL-1 antibodies in blocking buffer overnight at 4 . Following three washes with PBST, cells were incubated with FITC-conjugated biotinylated anti-rabbit IgG and℃ Texas Red-conjugated biotinylated anti-Goat IgG secondary antibodies, counterstained with DAPI, and then mounted. Fluorescence was observed using an Fig 2. Confirmed binding affinity between the CNTNAP4 Olympus BX51 microscope (200X) and Leica Confocal phages and rhNELL-1 by Kd ELISA. A. By increasing the microscope (1000X). number of phages using to incubate with rhNELL-1 pre-coated 7. Osteogenic differentiation assays ELISA plate, CNTNAP4 phage shows higher binding affinity For osteogenic differentiation, MC3T3 and stable than control phage. *: p<0.05 when compared to control phage. B. CNTNAP4 phage shows high binding affinity to full Cntnap4 KD cells were seeded on 24-well plates for ALP, length NELL-1 but not NELL-1 short isoform. *: p<0.05 when Alizarin Red staining, and Immunocytochemistry, on 6- compared to control phage. well plates for osteogenic genes expression test by realtime-PCR. Cells were treated with osteogenic differentiation medium (αMEM, 10% FBS, 50µg/ml ascorbic acid, 10mM β-glycerophosphate) with or without 500ng/ml rhNELL-1 or 100ng/ml BMP-2. ALP staining and Alizarin Red staining were performed as previously described (Shen et al., 2012). 8. MAPK activation assays by western blot Sub-confluent control and Cntnap4 KD MC3T3 were subjected to serum starvation for 18 hr, and then treated with PBS or rhNELL-1 (500ng/ml) for 10 min, or 30 min at 37 . Protein isolation and western blot were performed as previously described (Zhang et al., 2011b). ℃ Results 1. Identification and confirmation of CNTNAP4 as a cell surface protein binding to NELL-1 After sequencing, 22 of rhNELL-1 binding candidates were obtained (Table 2). The phages containing partial amino acid sequence of CNTNAP4 showed high binding ability to full-length rhNELL-1 and low binding ability to short rhNELL-1 isoform (Pang et al., 2014) by binding Kd ELISA (Fig 2). Further, both pull-down and Co-IP confirmed the physical interaction between NELL-1 and CNTNAP4 in MC3T3 and NMCC which had highest expression level of Cntnap4 among 12 types of cell lines and primary cells being screened (Fig 3). Phenotypically, high level of plasma membrane staining of CNTNAP4 was detected in control MC3T3 in contrast to barely detectable expression in stable Fig 3. Confirmed the binding between CNTNAP4 and NELL-1 proteins by pull-down and Co-immunoprecipitaion in MC3T3 Cntnap4 KD cells (Fig 4A). Significantly, there is and NMCC. A. among 8 types of tested cell lines, MC3T3 has the much higher NELL-1 staining intensity in control highest expression level of Cntnap4. B. among 4 types of tested MC3T3 than Cntnap4 KD cells after 30 min incubation primary cells, NMCC has the highest expression level of Cntnap4. with rhNELL-1, which indicates rhNELL-1 binding to NMCC: newborn mouse calvaria cells. mRC: mouse rib chondrocytes. hBMSC: human bone marrow stem cells. hARC: the cell surface specifically through CNTNAP4 (Fig human articular chondrocytes. C. Pull-down assay was performed 4A). The co-localization of CNTNAP4 and NELL-1 with MC3T3 and NMCC. More CNTNAP4 was detected when was further evaluated using confocal microscopy. coated beads with his-NELL-1. D. Co-IP assay was performed Immunofluorescent staining presented that majority of with MC3T3 and NMCC. More CNTNAP4 was detected when CNTNAP4 located on the plasma membrane, and the added his-NELL-1 to the cells. No bands were detected when not coated the agrose beads with anti-NELL-1 antibody. higher staining intensity of NELL-1 was found predominantly on the cell membrane after addition of exogenous rhNELL-1 although the intensity varies among stained cells (Fig 4B). This is indicative of formation of a binding complex of these two proteins on cell surface. 2. CNTNAP4 is indispensable for NELL-1’s osteogenic effect ALP staining at day 9 and Alizarin Red staining at day 14 revealed that rhNELL-1 can significantly induce osteogenesis and mineralization with control MC3T3, while these osteogenic effects of rhNELL-1 were completely abrogated by Cntnap4 KD. In contrast, the ALP and Fig 4. Confirmed the Co-localization of CNTNAP4 and NELL-1 proteins by Alizarin Red staining of BMP-2 immunocytochemistry in MC3T3. A. In control shRNA transfected MC3T3 cells, high level of plasma membrane staining of CNTNAP4 was detected. 30min after rhNELL-1 treated Cntnap4 KD MC3T3 stimulation, more NELL-1 was detected. In stable Cntnap4 KD cells, CNTNAP4 is at showed similar intensity to that of barely detectable expression level, and no much NELL-1 staining was detected even control MC3T3 indicating the after rhNELL-1 stimulation. B. In the exogenous rhNELL-1 added control MC3T3, dispensable feature of CNTNAP4 majority of CNTNAP4 located on the plasma membrane, and the higher staining intensity of NELL-1 was found predominantly in the cell membrane after addition of exogenous NELL-1. for BMP-2’s osteogenic effects (Fig 5A). At molecular level, we detected a time-dependent increase of Ocn and Opn staining in PBS and rhNELL-1 treated control MC3T3, while for each time point, rhNELL-1 treated group showed more staining than PBS treated group. For the Cntnap4 KD MC3T3, neither PBS nor rhNELL-1 group showed positive staining of Ocn or Opn (Fig 5B). The same trends can be found for the osteogenic markers mRNA expression. In the Cntnap4 KD MC3T3, there were neither significant changes of all the osteogenic markers as time advanced, nor differences between PBS group and rhNELL-1 group (Fig 5C). But BMP-2’s upregulation of these osteogenic markers, similar to ALP and Alizarin Red staining, was not affected (data not shown). Thus, CNTNAP4 is indispensable for NELL-1’s osteogenic effect. 3. CNTNAP4 is a specific receptor of NELL-1 in modulating osteogenesis Our previously published data shows that NELL-1 induced osteogenic differentiation and mineralization by activating MAPK signaling pathway as one of its mechanisms (Bokui et al., 2008; Zhang et al., 2011b). Assuming CNTNAP4 was a functional and specific receptor of NELL-1, it would be expected to elicit MAPK signaling upon its binding to NELL- 1, which is similar to the most of Fig 5. CNTNAP4 is indispensable for NELL-1’s osteogenic effect. A. ALP staining at day 9 and Alizarin Red staining at day 14 showed high staining intensity in the NELL-1 receptor-ligand interactions (Shi and BMP-2 groups of control shRNA transfected MC3T3, while in Cntnap4 KD MC3T3, and Massague, 2003). To address high staining intensity of ALP and Alizarin Red could only be found in BMP-2 group. B. A if this MAPK activation by rhNELL- time-dependent increase of Ocn and Opn staining in PBS and rhNELL-1 treated control 1 was through CNTNAP4, protein MC3T3 was detected, while for each time point, rhNELL-1 treated group showed more was isolated from control MC3T3 staining than PBS treated group. For the Cntnap4 KD MC3T3, neither PBS group nor rhNELL-1 group showed positive staining of Ocn or Opn. C. In control MC3T3, Alp, and Cntnap4 KD MC3T3 10 min Collagen Iα1 and Collagen Iα2 reached expression peak level 9 days after stimulation; and 30 min after rhNELL-1 Ocn, Opn and Bsp displayed a time-dependent increase pattern in control MC3T3, while stimulation, and high higher expression levels were detected in rhNELL-1 group than PBS group. In the phosphorylation levels of ERK and Cntnap4 KD MC3T3, there were neither significant changes of all the osteogenic markers as time advanced, nor differences between PBS group and rhNELL-1 group. *: p<0.05 JNK were detected as early as 10 when compared to control group. min after rhNELL-1 stimulation in the control MC3T3 as what we have shown before (Zhang et al., 2011b). Distinctively, there was no difference detected in the changes of pERK or pJNK level between PBS and rhNELL-1 treated Cntnap4 KD MC3T3 at all time points (Fig 6). This data implicates that exogenous NELL-1 activates MAPK signaling pathway to promote MC3T3 osteogenic differentiation through binding to CNTNAP4 as its specific receptor. Discussion Nell-1 is a secretory factor (Kuroda et al., 1999) with relatively high specificity in promoting bone regeneration (Zhang et al., 2010). NELL-1 has been proposed to act as an ECM component (Hasebe et al., 2012b), and transduce osteogenic signals through Ras-mitogen-activated protein kinase (MAPK) signaling (Bokui et al., 2008; Zhang et al., 2011b), and modulate Runx2 activity (Zhang et al., 2011b). But to date, the exact mode of action of NELL- 1, particularly if NELL-1 has specific receptor(s) on cell surface through which activating signaling remains unknown. Fig 6. CNTNAP4 knockdown blocked the activation of MAPK signaling by NELL-1 stimulation. In control MC3T3 (upper), significantly higher levels of In this study, we successfully pERK and pJNK were detected 10 min after rhNELL-1 stimulation. In Cntnap4 identified and validated Contactin KD MC3T3 (lower), NELL-1 stimulation didn't alter the expression level of pERK associated protein-like 4 (CNTNAP4) as a and pJNK. P: PBS, N: rhNELL-1. specific cell surface membrane receptor of NELL-1 in promoting osteogenesis. To our surprise, the Cntnap4 knockdown is not only responsible to rhNELL-1 induced osteogenesis, but may be also crucial to induce or maintain osteoblastic differentiation of preosteoblastic cells by endogenous Nell-1. The latter notion was derived from the lack or much lower level of elevated osteogenic markers in Cntnap4 KD MC3T3 cells treated with PBS Fig 7. Structure and potential binding sites of NELL-1 and CNTNAP4. NELL-1 is a as compared to control MC3T3 secreted protein comprised of 810 amino acids with a molecular weight of about 90kDa treated with PBS. To confirm the before N-glycosylation and oligomerization, it contains several structural motifs including an N-terminal thrombospondin-1-like (TSPN) domain (overlapping with a laminin G loss of rhNELL-1’s osteoinductive domain), coiled-coil (CC) domain, four von Willebrand factor type C (vWC) domains and effects was due to no specific six epidermal growth factor (EGF)-like domains (Zhang et al., 2010). Contactin binding site of rhNELL-1 after associated protein-like 4 (CNTNAP4), also known as Caspr4, is a transmembrane protein Cntnap4 knockdown, but not the that has a length of 1310 amino acids consist of a large extracellular domain, a single intrinsic defects possibly created membrane-spanning domain and a short cytoplasmic region at its carboxy-terminus. The extracellular region is composed of discoidin and fibrinogen-like domains, two EGF during the selection of shRNA repeats and four Laminin G domains, and the cytoplasmic region contains a binding site tranfected cells, we used a potent for PDZ domains (Spiegel et al., 2002). By analyzing the sequence of CNTNAP4 inserted and mechanistically different into the T7 phage, and the differences between full-length and short isoform of NELL-1, osteogenic protein from NELL-1, we speculated that the N-terminal of NELL-1, specifically TSPN domain, probably binds to the LamG domains of CNTNAP4 on the cell surface. The potential binding sites of BMP-2, on same cells for NELL-1 and CNTNAP4 were highlighted by blue dashed line. TM: transmembrane. osteogenic induction. As expected, BMP-2 was capable of inducing both Cntnap4 knockdown and control MC3T3 osteogenic differentiation without significant differences. This further validated that, CNTNAP4 functions as a specific receptor of NELL-1 to mediate its osteoinductive effects. This finding is a substantial progress of the ongoing basic and translational studies on NELL-1. The sequence of CNTNAP4 inserted into the T7 phage is located in the LamG domains on the extracellular part of CNTNAP4. Interestingly, the CNTNAP4 T7 phage didn’t bind to the short NELL-1 isoform (Pang et al., 2014) but has high binding affinity to full-length NELL-1. This may suggest that the N-terminal of NELL-1, specifically TSPN domain, probably binds to the LamG domains of CNTNAP4 on the cell surface to activate downstream signaling cascade including MAPK (Fig. 7). More tedious work of mapping the precise binding site/domain of NELL-1 and CNTNAP4 warrants to be carried out. Up to date, CNTNAP4 has been demonstrated to be highly expressed in central neural system (Spiegel et al., 2002), involve in neuron-glia interaction (Charles et al., 2002; Einheber et al., 1997) and interact with ligand of Numb protein X2 to modulate the proliferation and neuronal differentiation of mouse neural progenitor cells (Yin et al., 2014). CNTNAP4 gene has also been identified as a novel susceptibility gene of Autism spectrum disorders (ASDs) (O'Roak et al., 2012; Wang et al., 2010), while Cntnap4 deficient mice exhibited autism like behaviors (Karayannis et al., 2014). Significantly, we screened 12 types of non-neuron and glial cells, and found relatively high expression of Cntnap4 in MC3T3 and primary mouse calvaria cells. This opens a new revenue for the exploration of potential CNTNAP4 biological roles outside of the neural system. The identification of CNTNAP4 as a specific receptor of an osteogenic protein ligand - NELL-1 is a very critical step forward for the continuous research efforts in elucidating mode of action of NELL-1. References Bokui N, Otani T, Igarashi K, Kaku J, Oda M, Nagaoka T et al. (2008). Involvement of MAPK signaling molecules and Runx2 in the NELL1-induced osteoblastic differentiation. FEBS letters 582(2):365-371.

Charles P, Tait S, Faivre-Sarrailh C, Barbin G, Gunn-Moore F, Denisenko-Nehrbass N et al. (2002). Neurofascin is a glial receptor for the paranodin/Caspr-contactin axonal complex at the axoglial junction. Current biology : CB 12(3):217-220.

Cowan CM, Zhang X, James AW, Kim TM, Sun N, Wu B et al. (2012). NELL-1 increases pre-osteoblast mineralization using both phosphate transporter Pit1 and Pit2. Biochemical and biophysical research communications 422(3):351-357.

Desai J, Shannon ME, Johnson MD, Ruff DW, Hughes LA, Kerley MK et al. (2006). Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects. Hum Mol Genet 15(8):1329-1341.

Einheber S, Zanazzi G, Ching W, Scherer S, Milner TA, Peles E et al. (1997). The axonal membrane protein Caspr, a homologue of neurexin IV, is a component of the septate-like paranodal junctions that assemble during myelination. The Journal of cell biology 139(6):1495-1506.

Goessler UR, Bugert P, Bieback K, Stern-Straeter J, Bran G, Hormann K et al. (2008). Integrin expression in stem cells from bone marrow and adipose tissue during chondrogenic differentiation. International journal of molecular medicine 21(3):271-279.

Hasebe A, Nakamura Y, Tashima H, Takahashi K, Iijima M, Yoshimoto N et al. (2012a). The C-terminal region of NELL1 mediates osteoblastic cell adhesion through integrin alpha3beta1. FEBS letters 586(16):2500-2506.

Hasebe A, Tashima H, Ide T, Iijima M, Yoshimoto N, Ting K et al. (2012b). Efficient production and characterization of recombinant human NELL1 protein in human embryonic kidney 293-F cells. Molecular biotechnology 51(1):58-66.

Karayannis T, Au E, Patel JC, Kruglikov I, Markx S, Delorme R et al. (2014). Cntnap4 differentially contributes to GABAergic and dopaminergic synaptic transmission. Nature 511(7508):236-240.

Kuroda S, Oyasu M, Kawakami M, Kanayama N, Tanizawa K, Saito N et al. (1999). Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2. Biochemical and biophysical research communications 265(1):79-86.

Kwak J, Zara JN, Chiang M, Ngo R, Shen J, James AW et al. (2013). NELL-1 injection maintains long-bone quantity and quality in an ovariectomy-induced osteoporotic senile rat model. Tissue engineering Part A 19(3-4):426-436.

Nakamura R, Nakamoto C, Obama H, Durward E, Nakamoto M (2012). Structure-function analysis of Nel, a thrombospondin-1-like glycoprotein involved in neural development and functions. The Journal of biological chemistry 287(5):3282-3291.

O'Roak BJ, Vives L, Girirajan S, Karakoc E, Krumm N, Coe BP et al. (2012). Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature 485(7397):246-250.

Pang S, Shen J, Liu Y, Chen F, Zheng Z, James AW et al. (2014). Proliferation and osteogenic differentiation of mesenchymal stem cells induced by a short isoform of NELL-1. Stem cells.

Shen J, James AW, Chung J, Lee K, Zhang JB, Ho S et al. (2012). NELL-1 promotes cell adhesion and differentiation via Integrinbeta1. Journal of cellular biochemistry 113(12):3620-3628.

Shi Y, Massague J (2003). Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113(6):685-700.

Spiegel I, Salomon D, Erne B, Schaeren-Wiemers N, Peles E (2002). Caspr3 and caspr4, two novel members of the caspr family are expressed in the nervous system and interact with PDZ domains. Molecular and cellular neurosciences 20(2):283-297.

Ting K, Vastardis H, Mulliken JB, Soo C, Tieu A, Do H et al. (1999). Human NELL-1 expressed in unilateral coronal synostosis. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 14(1):80-89.

Wang LS, Hranilovic D, Wang K, Lindquist IE, Yurcaba L, Petkovic ZB et al. (2010). Population-based study of genetic variation in individuals with autism spectrum disorders from Croatia. BMC medical genetics 11(134.

Yin FT, Futagawa T, Li D, Ma YX, Lu MH, Lu L et al. (2014). Contactin-Associated Protein 4 Interaction with Ligand of Numb Protein X2 Modulates the Proliferation and Neuronal Differentiation of Mouse Neural Progenitor Cells. Stem cells and development.

Zhang X, Kuroda S, Carpenter D, Nishimura I, Soo C, Moats R et al. (2002). Craniosynostosis in transgenic mice overexpressing Nell-1. The Journal of clinical investigation 110(6):861-870.

Zhang X, Zara J, Siu RK, Ting K, Soo C (2010). The role of NELL-1, a growth factor associated with craniosynostosis, in promoting bone regeneration. Journal of dental research 89(9):865-878.

Zhang X, Peault B, Chen W, Li W, Corselli M, James AW et al. (2011a). The Nell-1 growth factor stimulates bone formation by purified human perivascular cells. Tissue engineering Part A 17(19-20):2497-2509.

Zhang X, Ting K, Bessette CM, Culiat CT, Sung SJ, Lee H et al. (2011b). Nell-1, a key functional mediator of Runx2, partially rescues calvarial defects in Runx2(+/-) mice. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 26(4):777-791.

Zhang X, Ting K, Pathmanathan D, Ko T, Chen W, Chen F et al. (2012). Calvarial cleidocraniodysplasia-like defects with ENU-induced Nell-1 deficiency. The Journal of craniofacial surgery 23(1):61-66.

Zou X, Shen J, Chen F, Ting K, Zheng Z, Pang S et al. (2011). NELL-1 binds to APR3 affecting human osteoblast proliferation and differentiation. FEBS letters 585(15):2410-2418.